G-protein-coupled receptors (GPCRs) are the targets of a large fraction of all clinically useful drugs and many drugs of abuse. GPCRs often desensitize in the continued presence of an agonist, and physiological responses produced by these receptors are thus diminished. It is thought that desensitization underlies drug tolerance, whereas signals produced by receptors that do not desensitize may lead to the adaptive changes that underlie drug dependence. It is therefore important to understand the factors that influence GPCR desensitization in neurons. Since desensitization can vary greatly depending on the type of cell studied, it is also essential to study this process in native neurons. Using an in vitro model of GPCR desensitization, we have found that adenosine A[1] receptors (A1Rs) located on presynaptic terminals desensitize much more slowly than the same receptors located on postsynaptic dendrites in the same neurons. Therefore, we hypothesize is that desensitization of neuronal GPCRs can be influenced by subcellular location, and that presynaptic terminals are sites where GPCRs are resistant to desensitization. The work proposed here will determine if desensitization of presynaptic and/or postsynaptic adenosine A1Rs is mediated by the conserved (GRK/arrestin) mechanism responsible for desensitization of GPCRs in non-neuronal cells (Specific Aims 1 and 2), and will determine if GPCRs in presynaptic terminals desensitize slowly as a rule (Specific Aim 3). We will study native neuronal receptors using electrophysiology and radioligand binding techniques, and will use viral vectors to perturb endogenous regulatory pathways. We will also use non-viral transfection and viral vectors to express A1Rs and mutant A1Rs in cerebellar granule neurons and A1R-deficient neurons, in an attempt to determine the mechanism(s) whereby these receptors desensitize. The results of these experiments will provide important new information about GPCR desensitization in neurons. If our hypothesis is correct, we will identify presynaptic terminals as key sites for the adaptive changes responsible for the development of drug effects, drug tolerance and drug dependence.